The purpose of this study was to determine the role of TRPV1 receptor in modulating neuronal activity of the dorsolateral periaqueductal gray (dl-PAG) through excitatory and inhibitory synaptic inputs. First, whole cell voltage-clamp recording was performed to obtain the spontaneous miniature excitatory postsynaptic currents (mEPSCs) and inhibitory postsynaptic currents (mIPSCs) of the dl-PAG neurons. As 1 µM of capsaicin was applied into the perfusion chamber, the frequency of mEPSCs was increased from 3.21±0.49 to 5.64±0.64 Hz (P<0.05, n=12) without altering the amplitude and the decay time constant of mEPSCs. In contrast, capsaicin had no distinct effect on mIPSCs. A specific TRPV1 receptor antagonist, iodo-resiniferatoxin (i-RTX, 300 nM) decreased the frequency of mEPSCs from 3.51±0.29 to 2.01±0.2 Hz (P<0.05, n=8) but did not alter the amplitude and decay time. In addition, i-RTX applied into the chamber abolished the effect of capsaicin on mEPSC of the dl-PAG. In another experiment, spontaneous action potential of the dl-PAG neurons was recorded using whole cell current-clamp methods. Capsaicin significantly elevated the discharge rate of the dl-PAG neurons from 3.03±0.38 to 5.96±0.87 Hz (n=8). The increased firing activity was abolished in the presence of glutamate NMDA and non-NMDA antagonists, 2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione. The results from the current study provide the first evidence indicating that activation of TRPV1 receptors increases the neuronal activity of the dl-PAG via selective potentiation of glutamatergic synaptic inputs.